Detoxification and decontamination using nanotechnology therapy

ABSTRACT

A method of removing a toxic compound comprising contacting the toxic compound with a particle having two regions, the first region containing a detoxifying enzyme and the second region containing a material selected to partition the toxic compound into the second region. The particle may be a nanoparticle.

BACKGROUND OF THE INVENTION

Drug toxicity in humans can result from causes such as therapeuticmisadventure, illicit drug ingestion, or suicide attempt. Drug toxicityis a major health care problem throughout the world and results insignificant financial costs as well as potential harm including possibledeath. Unfortunately, the vast majority of life-threatening drugintoxications do not have specific pharmacological antidotes toameliorate their physiological effects. Other than providing supportivetherapy to affected individuals, often little can be done to helpindividuals affected by most drug intoxications. Drug toxicity can alsobe a problem in veterinary medicine.

When a chemical, such as xenobiotic, is administered to an organism, twoevents must generally occur for a biological response to be triggered.First, the xenobiotic must be transported to the site of action (the“target site”). Second, after arriving at the target site, thexenobiotic must interact with the intended target in an appropriatemanner. Interaction of a xenobiotic with the target site is governedlargely by two factors:

a. the size and shape of the xenobiotic which controls how well thexenobiotic interacts with the target site; and

b. the nature and relative positions of appropriate functional groups ofthe xenobiotic which affects the type and strength of its interactionwith complementary groups of the receptor.

Many physicochemical properties can be used to model receptorinteraction. For example, molar volume (MV) is an overall measure ofmolecular size, while the energy of the lowest unoccupied molecularorbital (E_(LUMO)) is a crude measure of the electron-accepting abilityof a given chemical compound.

Several methods for treating various drug intoxications currently exist.Immunotoxicotherapy can produce purified drug-specific antibodies totreat some potentially fatal cases of drug poisoning. By linking thetoxic drug to albumin and using it as a hapten, high affinity antibodieswith excellent specificity can be theoretically formed for use against aparticular molecule or a class of molecules. The last several years havebrought major innovations in the safety and efficacy ofimmunotoxicotherapy. In addition, advances have occurred in theprocesses of fragmentation which permits a greater volume ofdistribution (VOD) and diminished risk of sensitization and enhancedrenal elimination.

Volume of distribution is defined as the volume of fluid that would benecessary to contain the amount of drug in the body at a uniformconcentration equal to that in plasma. Thus, the definition assumes thebody is a single homogeneous fluid compartment and the drug is evenlydistributed throughout. The VOD may exceed the actual volume of thebody. In application, VOD defines the concentration following oneintravenous dose and roughly describes tissue penetration. A large VODindicates good tissue penetration, while a small VOD indicates poortissue penetration.

The binding portion of the antibody, known as the Fab fragment, has beenfound to reduce the physiological effects of drugs, such as digoxin,PCP, cocaine, colchicine, and tricyclic antidepressants in variousanimal models. However, despite these reported advances and thepurported potential advantages of antibody use over other drugintoxication therapies, only one commercial antibody product iscurrently available to treat drug overdoses in humans. Digibind®, adigoxin immune Fab, is a lyophilized powder of antigen binding fragments(Fab) derived from specific antibodies raised from sheep. It has beenshown to be highly effective in treating the life threateningcardiotoxic effects of digoxin, an inhibitor of the myocardial Na⁺/H⁺ATPase pump.

A number of reasons explain the lack of widespread immunotoxicotherapyuse in humans. First, there is no guarantee that a specific antibody canbe produced that can effectively bind to a given target toxic drugmolecule or its associated class of molecules. In addition, sinceantibodies are directed at specific chemical moieties, antibodies cannotoffer broad substrate detoxification to an entire class of intoxicants.

Moreover, the ability and capacity of antibodies to effectively binddrug toxins is severely limited by each antibody's stoichiometry. OneFab fragment can typically bind only one target molecule. This propertylimits the binding effectiveness of these antibodies, especially fordrug toxicities that typically include large doses and extensive tissueand plasma protein binding (e.g., amiodarone).

Immunotoxicotherapy has also been shown to be only effective insituations where the amount of toxic drug in the bloodstream isrelatively small and protein and tissue binding is not significant. Anexample of a drug suitable for immunotoxicotherapy is the drug digoxin.The plasma concentration of digoxin that begins to cause cardiactoxicity is approximately 1.7 ng/ml, with only approximately 25±5% of itbeing bound in plasma. In contrast, the same toxic plasma concentrationsfor the drugs amiodarone and amitriptyline begin at approximately 3,500ng/ml and 1,000 ng/ml, respectively, corresponding to 99.98±0.01% and94.8±0.8% drug binding in plasma, respectively. This fact largelyexplains why antibodies are more effective against drugs such as digoxinwhich cause toxicity at low concentrations (approximately 1 nM).

In addition, most antibodies that have been produced have been formedfor use against toxic drug molecules in humans were isolated fromanimals (e.g. Digibind® is produced from sheep antibodies). Therefore,there remains a potential risk of allergy and aphylactic shock, becausethe Fc portion of the antibody, which is the most antigenic portion ofthe antibody, is cleaved from the binding portion of the immunoglobulin(Fab fragment) using papain. Also, unless major advances are made in themolecular production of antibodies, particularly human monoclonalantibodies, the quantity of antibodies which can be formed is verylimited. Consequently, it is extremely expensive and impractical tomanufacture the large quantities of antibodies required to treat drugtoxicities involving drugs that are highly protein and tissue bound aswell as toxicities arising from drugs having relatively high toxic bloodconcentrations.

Even for a drug like digoxin that causes drug toxicity at much lowerconcentrations than do drugs such as amiodarone and amitriptyline and ismuch less protein bound, the cost of treatment can still be prohibitive.For example, depending upon the blood concentrations of digoxin, 10-20vials or more of Digoxin immune Fab (ovine), trade named Digibind®, maybe required to effectively treat a 70 kg (155 pound) individual. Thiscan translate to a cost of approximately $10,000 to $30,000.Accordingly, the cost of using an agent such as Digibind® can beprohibitively expensive where the potency of the drug is much lower andconsequently a much greater amount of toxic drug (e.g., amiodarone oramitriptyline) would be required to be bound by the antibody.

Therefore, in the current state of development, antibodies are not aviable option to treat the vast majority of drug overdoses. Furthermore,even if new advances in molecular medicine allowed human-derivedantibodies to be produced in mass quantities at a reasonable cost, theirslow onset time of actions in humans is another limiting factor. Forexample, the median time to initial response for Digibind® is reportedto be 18 minutes. Only 75% of patients showed evidence of responsewithin 60 minutes. Slow response combined with inability to providebroad substrate detoxification and high cost are expected to continue tomake antibodies a poor choice for the treatment of drug toxicity.

Another method for reducing toxic drug effects is infusion of enzymesinto the blood. This method may be feasible to ameliorate toxic in-vivoeffects of drugs. Specifically, the acute physiological effects ofcocaine in various animal species have been reported to be acutelyameliorated using animal and human butyrylcholinesterase, the principalesterase in the blood that degrades cocaine to its major metabolites.

While sometimes effective, this approach suffers from a number ofdrawbacks. First, water-soluble plasma enzymes, such asbutyrylcholinesterase, do not metabolize the majority of drugs. Second,infusion of enzymes directly into the blood does not permit thebeneficial and synergistic actions of initially partitioning the drug athigh local concentrations in an environment containing highconcentrations of an enzyme. The reaction velocity (V) of enzymesgenerally obey Michaelis-Menten kinetics. Thus, although V isproportional to the substrate concentration at low concentrations (firstorder) the reaction velocity is constant at higher substrateconcentrations (zero order). Zero order kinetics largely negates thepotential treatment advantage derived from having a high local enzymeconcentration.

Although the infusion of water soluble enzymes, such asbutyrylcholinesterase, into the blood can cause rapid and efficientconversion of ester-type drugs, this approach is not applicable for P450microsomal fractions. P450 microsomal fractions require a lipidenvironment and are extensively integrated into the cellular membranesof hepatocytes and other cells.

Another method for detoxifying drugs is hemodialysis. Hemodialysis canbe used to detoxify the blood stream from various drugs and metabolicdisorders using hemoperfusion and hemodialysis. However, for a number ofreasons, this method is not appropriate for the vast majority of lifethreatening drug overdoses.

First, many drugs that cause toxicity in humans are not susceptible toremoval from the body by hemodialysis, either due to theirphysicochemical properties or their large volumes of distribution. Forexample, despite being primarily renally excreted (approximately 65% ofan administered oral dose), digoxin cannot be effectively dialyzed,whereas amiodarone cannot be effectively removed from the blood due to aVOD of approximately 66 L/kg. This large VOD means that most amiodaroneis sequestered in compartments other than the intravascular space. Inthe latter case, hemodialysis would be futile because the volume ofblood required to circulate through a dialysis machine would be toolarge to be practical.

Second, the rate of removal of a drug from the blood must be taken intoaccount. Hemodialysis and hemoperfusion are slow (e.g., generally hours)to generally remove drugs at toxic levels from the bloodstream.

For example, the intoxications of two testbed drugs, amiodarone andamitriptyline, are frequently life threatening because of their severeeffects on cardiac function. Accordingly, treatment under theseconditions must be initiated immediately or a patient may die.Consequently, hemodialysis or hemoperfusion cannot be used as atreatment for fast acting life threatening drugs such as amiodarone andamitriptyline. In addition, these approaches require the placement oflarge arterial and venous cannula prior to circulation of blood throughthe dialysis machine. As a result of its shortcomings, hemodialysis hasminimal applications in treating drug toxicity. However, hemodialysismay be applicable for toxic drugs with low volumes of distribution andfor those toxins that do not immediately produce life threateningeffects.

Another method for treating drug poisonings is the use of specificpharmacological antidotes. However, of the many types of drug poisoningsin humans, only a few have identified specific pharmacologicalantagonists that can be used to quickly and selectively reverse theirdeleterious physiological effects. Probably the best two examples ofeffective pharmacological antidotes are the muscarinic-cholinergic andnarcotic receptor antagonists, atropine and naloxone, respectively.Atropine blocks the physiological effects of excessive acetylcholinelevels on muscarinic receptors. Therefore, it is effective againstorganophosphate-based insecticides as well as nerve gas agents.

In an analogous manner, naloxone blocks most of the physiologicaleffects (e.g., respiratory depression) of narcotic overdosage.Therefore, it is effective in reversing the physiological effects ofpotent narcotics such as heroin and fentanyl. Although receptorantagonists are highly efficacious, rapid and specific in reversingthese types of life threatening drug poisonings, they do so bypreventing access of the agonist to its cellular locus of action (i.e.,the receptor). Receptor antagonists neither alter the free blood drugconcentration, nor promote its biotransformation to less toxicmetabolites and its ultimate excretion from the body. This is clinicallyimportant, because deaths have been reported when the biological effectsof a receptor antagonist outlives that of the drug toxin.

In contrast to the above situations where life saving measuresfrequently must be instituted immediately or death may occur, adifferent type of pharmacological antidote, a biochemical one, may alsobe used to treat drug toxicity problems that are less emergent innature. The best-known example of this type of treatment is usingN-acetylcysteine to replenish hepatic stores of glutathione in thesetting of acetaminophen (Tylenol®) overdosage.

However, high levels of acetaminophen in the blood deplete liversulfhydryl stores. This, in turn, allows the formation of a highlyreactive intermediate, N-acetyl-benzoquinoneimine, that can cause freeradical injury to the liver. Acetaminophen toxicity is a slow ongoingprocess which develops over a period of several hours to days.Accordingly, it is not necessary to treat acetaminophen toxicity with amethod which quickly decreases the chemical level to save lives. Aneffective treatment to acetaminophen overdosage is already available. Byreplenishing hepatic stores of glutathione using N-acetylcysteine orallyor intravenously, the liver can be protected against further injury fromtoxic metabolites of acetaminophen. However, biochemical antidotes canonly be used to treat the narrow class of drugs which result in slowacting toxicity.

Thus, based on the lack of effectiveness of currently availabletreatments for most drug intoxications, there is a need for new andimproved technologies for rapidly and inexpensively reducing the freedrug concentration for a wide variety of drugs. Drugs to be treated maybe introduced in potentially toxic levels into living organisms as wellas onto non-biological surfaces or bodies such as metal or wood.

SUMMARY

A method for removal of at least one target chemical from a regionincludes the steps of adding a nanoparticle size bioparticle to theregion and partitioning at least a portion of the target chemical intoor onto the bioparticle. The method results in reducing the activeconcentration of the target chemical. The region can be a solution.Partitioning can result from differences in physicochemical propertiesbetween the bioparticle and the target chemical and/or from adsorptionof the target chemical on a surface of the bioparticle.

A method for removal of at least one target chemical from a regionincludes the steps of adding a nanoparticle size bioparticle having atleast enzyme incorporated therein to the region and biotransforming atleast a portion of the target chemical into at least one substantiallyinactive metabolite. The region can be a solution. The enzyme canpreferably be a genetically cloned enzyme.

A method for removal of at least one target chemical from a regionincludes the steps of adding a bioparticle having at least one enzymeincorporated therein and partitioning at least a portion of the at leastone target chemical into or onto the bioparticle. The method results ina portion of the target chemicals being transformed into at least onesubstantially inactive metabolite. The region can be a solution. Thebioparticle can be a nanoparticle. In one embodiment, the nanoparticlecan include a silica nanotube having an alkyl compound attached to thesilica nanotube. The bioparticle can have a size from approximately 1 to100 nm or preferably from approximately 1 to 5 nm. Enzymes can includegenetically cloned enzymes.

A method for treating a patient exposed to a toxic drug includes thesteps of providing a plurality of nanosized bioparticles capable ofmitigating the effects of the toxic drug through at least one mechanismselected from the group consisting of partitioning the toxic drug intoor onto the bioparticle and transforming the toxic drug into at leastone substantially inactive metabolite, and introducing the plurality ofbioparticles and then introduced into the patient. This method can alsobe applied to animals in the practice of veterinary medicine.

A composition for detoxification includes a plurality of nanoparticles.The nanoparticles have at least one surface adapted for toxic drugattachment. The nanoparticles are selected from the group consisting ofmicroemulsions with nanoscale oil cores having soft surface films,hydrophobic cores having porous or soft shells and hard surfaces forselective binding of toxins. The nanoparticles can include attachedenzymes for chemically degrading toxins. Preferably, the attachedenzymes include genetically cloned enzymes.

DETAILED DESCRIPTION OF THE INVENTION

Bioparticles have been discovered which provide effective and generallycomplementary mechanisms for reducing the free concentration andbiological effects of a broad range of toxins including drugs. Recentadvances in particle science engineering combined with developingmolecular medicine have been found to provide highly effectivetherapeutic strategies aimed at effectively treating a wide variety oftoxic poisonings, including drug poisonings. Previously, applicationsfor particle technology were limited by the size of available particles.For example, particles available for use had been too large to beeffectively used with living organisms.

With a reduction in size of available particles to less than 100 nm, oreven as small as typical molecular dimensions (1-5 nm), the inventionhas created bioparticles having nanoparticle sizes which are smallenough to effectively interact with living organisms. It is noted thatmolecules may be less than 1 nm in diameter or larger than 5 nm indiameter. For example, proteins which are polymers or macromolecules canbe 5 nm or larger in diameter. As used herein, the term nanoparticlesrefer to particles having sizes less than approximately 100 nm. Thesebioparticles can act to bind with targeted drugs and other toxicsubstances and/or to quickly degrade these drugs and toxins into largelyinactive reaction products. The invention can have valuable applicationsfor a wide variety of uses such as drug detoxification (e.g., drugoverdose), military (e.g., toxic warfare agents), industrial (e.g.,manufacturing processes), environmental (chemical spill clean up), aswell as many other purposes.

Drug poisonings may be treated by bioparticles which can use any or allof the following mechanisms:

1) partitioning a targeted drug onto the bioparticles by exploitingdifferences in physicochemical properties and/or using moleculartemplating to adsorb the drug onto functionalized surfaces ofbioparticles;

2) biotransforming a targeted drug into an inactive metabolite(s). Forexample, an enzyme, such as a human purified and genetically cloned highactivity enzyme, may be incorporated into a bioparticle to providebiotransformation effects on a targeted drug or toxin, or

3) preferably, providing a bioparticle which combines both approaches(1) and (2).

These above methods are not limited to oral or intravenous use, butcould also be employed to remove toxins from biological surfaces such asskin or non-biological surfaces such as metal, wood or plastic.Moreover, the above methods can be used to detoxify a broad range oftoxins.

The invention allows the synthesis of bioparticles that can directlyreduce the free drug concentration in the blood, either by exclusivelypartitioning it inside the bioparticle, or more preferably, bypartitioning and biotransforming the drug into an inactive metabolitewithin the bioparticle and ultimately promoting excretion from the body.With this approach, the principles of lipid partitioning and/oradsorption (via molecular templating) may act in a highly synergisticmanner with those of biotransformation to provide drug detoxificationsystems with complementary detoxification mechanisms to provide addedeffectiveness. Thus, by providing a very high local concentration oftoxic drug (substrate), partitioning and/or adsorption can dramaticallyincrease the rate of enzymatic degradation depending upon the KM of theenzyme and its enzymatic efficiency in the bioparticle.

Nanoparticles (such as those shown in FIG. 1) have been created fortreating a broad range of toxins. For example, “soft particles” such asmicroemulsions with nanoscale oil cores and “soft/hard” particles withhydrophobic cores for lipophilic drugs and various shells each permitrelatively selective drug penetration. “Templated particles” allow fortoxins to be selectively fitted into pores in their surface generallywith 1 or 2 point binding of the toxin to the nanoparticle. The abovenanoparticle types can preferably also include an attached enzyme foreven more rapid degradation of the target drug and/or toxin.

Soft/hard particles are ones consisting of a core composed of a liquidor a (softer) polymeric organic material surrounded by or encapsulatedin a shell composed of an inorganic or (harder) polymeric organicmaterial. The example of a liquid encapsulated in a shell of metal oxideor metal carbonate can be prepared by making a solution of the metaloxide precursor in an oil and exposing droplets of the solution to wateror carbonate ion, respectively. This could be accomplished in aerosol ormicroemulsion apparatus. The example of a liquid encapsulated in a shellof organic polymer can be prepared by making a microemulsion stabilizedby surfactant having reactive vinyl functionality, followed byinitiating polymerization of the surfactant molecules around the surfaceof individual oil droplets in the system. The example of a (soft)polymeric organic core encapsulated in a (hard) polymeric shell can beprepared by dispersing a polymer gel in a solution of organic monomerprecursor to a highly crosslinked or crystalline polymer, followed byinitiation of polymerization of the dissolved monomer.

Templated particles are ones consisting of material, either as the coreor shell component, that incorporates molecularly-sized and shaped poresinto its structure. These materials can be prepared by including in thesynthesis mixture suitably derivatized molecules or mimics (thetemplates) of the chemicals (in this application the toxins) of interestto be captured by the bioparticles. After the particles are formed andcollected, the templating species are removed from the particles bydialysis or pyrolysis, leaving the desired pores as selective sites inwhich toxic molecules to be removed can be trapped. Examples are variousmetal oxides or solid organic polymers that have pores templated forneural amines, aromatic compounds or terpenes.

A shell containing pores can provide significant improve theeffectiveness of nanoparticle-mediated drug detoxification. There are anumber of potential design benefits of encapsulating either ahydrophilic or a hydrophobic environment with a solid shell containingnanopores. First, it could act to stabilize a nanoemulsion injected intothe blood to prevent a dilutional effect on emulsion function. Second,it could act as a highly selective molecular filter by allowing onlymolecules of certain physical dimensions access to the bioparticleinterior. Therefore, bioparticles could be targeted against at toxicmolecules with molecular weights (MWs) at or below the selected cutoffpoints of molecular size. Third, by “trapping” locally highconcentrations of coenzymes and cofactors important to CYP activity suchas oxidoreductase (MW=75,000) and cytochrome b5 (MW=17,500) within thebioparticle interior and preventing their escape, a bioparticle with ashell incorporating nanopores would potentially enhance (or at leasthelp preserve) optimal P450 enzymatic activity.

The reconstitution of P450 enzymes, especially the CYP 3A4 fraction, maybe technically challenging with regards to preserving its catalyticactivity, especially in blood. Inclusion of a shell can greatly aid thisprocess. Fourth, a bioparticle shell with nanopores could preventproteolytic degradation of CYP fractions while in the blood (i.e., armorthe enzymes). It will allow ingress of smaller sized toxic drugmolecules and easy egress of more water-soluble metabolites, but excludethose molecules greater than the pore size cutoff. In contrast, CYPfractions incorporated into soft bioparticles may not only besusceptible to degradation in blood, but their local concentrations ofcofactors and coenzymes may not be preserved (see the second point givenabove). Fifth, a solid shell with nanopores could be designed to providea biodegradable platform where the functional activity of thebioparticle is determined by the blood half-life of biodegradable shell.That is, once the job of biotransformation is complete, thebiodegradable particle can slowly disintegrate and releases the CYPfractions into the blood where they are degraded. Sixth, design of theshell and its surface characteristics (e.g., ionicity, lipophilicity)may be critical to prevent (or minimize) uptake into RBCs or thereticuloendothelial system (RES), or eliminate the risk of hemolysis.The goal of bioparticle design is to keep the bioparticles within thevascular compartment. Seventh, if the shell of the nanobioparticle issized small enough to be directly excreted via the kidney, it may bepossible to directly sequester a highly lipophilic drug within thebioparticle via partitioning and/or adsorption, or to attach it to evensmaller sized nanoparticles by adsorption onto its surface.

In a manner similar to what is observed clinically when the Fab antibodyDigibind® is used to treat digoxin toxicity, if the size of the“nanoparticulobody” (a bioparticle acting functionally like an antibody)is less than a 50,000-60,000 MW molecule, it may be directly excretedfrom the body by the kidneys, particularly if its surface has a neutralor positive charge. Using molecular templating, highly selectiveadsorption of various molecules (and members among their chemical class)can be achieved. In this scenario, the P450 element of the bioparticlewould not be needed. In essence, the nanoparticle is accomplishing PhaseI and II biotransformation without the actual need for enzymemodification. That is, it renders the toxic drug molecule more polar bycomplexing it to a nanoparticle, which in turn allows excretion from thebody. This type of adsorption would be highly selective and would mostlikely be applicable only to those molecules (or class of drugs sharinga common chemical moiety) it was originally designed for. In contrast,if a lipid partitioning system could be incorporated into a bioparticle,while keeping its size less than 50-60 kDa, it would be useful for mostlipophilic drugs.

One approach for preparing such nanoparticles entails using thetemplate-synthesis method. In this method, the pores in a microporousmembrane or other solid are used as templates to prepare thenanoparticle. The membranes employed contain cylindrical pores withmonodisperse diameters. The pore diameter can be controlled at will overa range from iess than 1 nm to as large as tens of micrometers. Acorrespondingly large range of nanoparticle sizes can be prepared. Thetemplate method is very versatile. It has been used to preparenanoparticles composed of metals, semiconductors, other inorganicmaterials, carbons, etc. Nearly any method used to prepare bulkmaterials can be adapted to allow for synthesis of nanoparticles withinthe pores of a microporous template membrane. Significantly, a hollowtubular nanostructures can be obtained.

Membranes that have been used to prepare nanoparticles via the templatemethod are shown in FIG. 3. These are microporous aluminas preparedelectrochemically from aluminum metal. The upper set of micrographsshows an in-house prepared membrane of this type. In this case the poresare approximately 60 nm in diameter. The lower micrograph in FIG. 3shows a commercially-available membrane of this type. In this case thepores are 200 nm in diameter. These micrographs illustrate the importantpoint discussed above that the pore diameter in such membranes (andcorrespondingly the diameter of the nanoparticle obtained) can becontrolled at will. For these microporous alumina template membranes,the pore diameter is varied by varying the potential used during theelectrochemical formation of the membrane.

Toxins can be adsorbed onto specialized nanoparticles to achieve drugdetoxification. Nanoemulsions have been synthesized for acutely reducingthe free concentration of potentially toxic molecules. FIG. 2 shows theeffect of Pluronic® L-44 Micelles and microemulsions in reducing theconcentration of the drug amitriptyline. A micelle may be defined as acolloidal aggregate of amphipathic (surfactant) molecules, which occursat a well-defined concentration known as the critical micelleconcentration. The typical number of aggregated molecules in a micelle(aggregation number) is from approximately 50 to 100.

Poloxamer, or Pluronic® gels are made from selected forms ofpolyoxyethylene-polyoxypropylene copolymers in concentrations rangingfrom 15 to 50 weight %. Poloxamers generally are white, waxy,free-flowing granules that are practically odorless and tasteless.Aqueous solutions of poloxamers are stable in the presence of acids,alkalis and metal ions. Commonly used poloxamers include the 124 (L-44grade) used above as well as 188 (F-68 grade), 237 (F-87 grade), 338(F-108 grade) and 407 (F-127 grade) types, which are freely soluble inwater. The trade name “Pluronic®” is used in the US by BASF Corp., ofMount Olive, N.J., for pharmaceutical and industrial grade poloxamers.

In the example shown in FIG. 2, a nanoemulsion was produced usingPluronic® L-44 (micelles) with or without addition of ethylbutyrateester. Ethylbutyrate ester was added at a low (ME-I) and high (ME-II)concentration. As seen in FIG. 2, the emulsion effectively sequesteredsignificant quantities of amitriptyline, a tricyclic antidepressantagent with potential cardiotoxic effects, when compared to the salinecontrol shown. The system labeled “Micelles” did not possess the oilcore of ethylbutyrate ester which was possessed by both ME-1 and ME-II.By comparing the Micelles system to ME-1 and ME-2, it can be seen thatthe oil core significantly increased the amount of amitripylineadsorbed, the higher oil concentration (ME-II) adsorbing significantlymore amitripyline than the lower oil concentration (ME-I). Drugs thatmay be amenable to this type of detoxification are not limited totricyclic antidepressant agents such as amitriptyline, but may includedrugs and toxins from all drug classes that have an affinity for, tendto combine with, or are capable of dissolving in lipids (lipophilicdrugs).

Many types of oils, surfactants, and cosurfactants may be used toproduce bioparticles based on nanoemulsion technology. The bioparticlecomposition can be varied depending on the goal of the therapy and thespecific properties of the target toxin. For example, a particle systemprepared for intravenous use would, be preferably varied in compositioncompared to a similar system prepared for skin or metal decontamination.

Another type of bioparticle that can be used to detoxify drugs can beprepared using the template-synthesis method. Template-synthesizednanoparticles can be either hollow nanotubules or solid nanoparticlesand they can remove the toxic substance by either adsorption on theirsurfaces or extraction into the hollow part of the nanotubule. Suchtemplate-synthesized nanoparticles can be composed of a wide variety ofmaterials including metals, polymers. semiconductors, other inorganicmaterials, carbons, etc. The size of these nanoparticles (both thediameter and the length) can be controlled at will from the nm regime tothe micrometer regime.

One embodiment of this technology is to use the template method toprepare hollow cylindrical silica nanoparticles. These nanoparticles arepreferably prepared by using sol-gel template synthesis of silica withinthe pores of a microporous alumina template membrane. The silica tubulesin this case were prepared using tetraethylorthosilicate as the startingmaterial; however, other precursors are available for preparing silicavia the sol-gel method. Sol-gel silica nanotubules of this type havebeen prepared in the pores of various microporous alumina template ofthe type shown in FIG. 3. It has been shown that both the inside andoutside diameters and the length of the silica nanostructures can becontrolled by varying the diameter of the pores and the thickness of thetemplate used.

The tubular silica nanostructures prepared in this way can bederivatized both on their outside and inside surfaces with chemicaland/or biochemical reagents. One approach for doing this is to usewell-known silane chemistry. Hundreds of silanes are availablecommercially, so this is a very versatile route for chemically andbiochemically derivatizing these silica nanostructures. This isimportant because such derivatization allows these nanotubules toextract or adsorb specific chemical reagents and allows them to catalyzespecific biochemical reactions. In addition because the inside andoutside of the tubules can be derivatized with different reagents, theinside and outside chemistry/biochemistry can be different. This isimportant because, for example, it might be desirable to have theinterior of the nanotubules hydrophobic so that they will extractspecific molecules but the outside hydrophilic so that the nanotubulescan be dispersed in an aqueous-based medium (e.g. blood).

Two embodiments of this concept are discussed herein. The firstembodiment derivatizes the inside surfaces of the silica nanocylinderswith a hydrophobic 18 carbon alkyl silane. In this case the outsidesurface is left underivatized so that the outside surface retains thehydrophilic character of silica. The second case entails derivatizingboth the inside and outside surfaces with trimethoxybutyl aldehyde tointroduce the aldehyde functionality to the surfaces. This aldehydefunctionality can then be reacted with terminal amino sites on a proteinmolecule to covalently attach the protein to the nanotubules. Attachmentof the protein glucose oxidase (GOD) can be performed in this way. Manyother proteins have been attached using this general method.Accordingly, it is a versatile way to attach proteins to the surfaces ofthese nanotubules.

Scanning electron micrographs of an alumina template membrane having aplurality of open pores which can be filled with silica nanotubes areshown in FIGS. 3(a)-3(c). FIG. 3(a) shows a surface image of an aluminatemplate membrane demonstrating a high packing density of pores, thepores having diameters of approximately 60 nm. FIG. 3(b) shows a crosssectional image of the template membrane shown in FIG. 3(a) while FIG.3(c) shows a perspective view of the template membrane. Although 60 nmnominal pore diameters are shown, pore diameters of the templatemembrane can be readily controlled.

In an embodiment of the invention, a long chain alkyl carbon (such asC₁₈) is added to silica nanotubes positioned within a template membranefor the purpose of adsorbing a target molecule. It is noted that thefield of protein and enzyme attachment to particles, otherwise referredto prolifically in the literature as “enzyme immobilization”, is amature science, and that the methodologies described in this portion ofthe application are similar to ones described earlier but that theselection of which enzymes used and the overall particle composition isunique.

The 18-carbon alkyl (C₁₈) silane was chosen because this renders theinsides of the nanotubules hydrophobic. The nanotubules with the C₁₈groups inside can then be used to extract hydrophobic molecules from acontacting solution phase. Again, in this case the outsides of thenanotubules remain hydrophilic silica and this allows these tubules tobe dispersed into solutions containing polar solvents. The most obviousexample is water, but the same principle applies for other polarsolvents. Obviously, the outside could also be derivatized with thehydrophobic silane and such tubules could then be dispersed intosolutions containing nonpolar solvents. Other alkyl silanes could beused to tune the extraction selectivity of the derivatized nanotubules.Examples include using shorter chain (e.g. C₈) alkyl silanes to make thetubules less hydrophobic on the inside, using aromatic silanes, usingsilanes with specific chemical functionalities (e.g., acidic or basic),etc.

The hydrophobic C₁₈ silane-containing tubules were used to extract ahydrophobic target molecule (7,8-benzoqunoline or BQ) from a diluteaqueous solution. Extraction was accomplished in two ways. In the firstmethod the hydrophobic nanotubules were left embedded within the poresof the template membrane, and a piece of the membrane was simplyimmersed into and then removed from the solution of the target molecule.Removing the membrane also accomplished the removal of the targetmolecule BQ sequestered inside. In the second method, the nanotubuieswere liberated from the template membrane, by dissolving the membrane inphosphoric acid solution. The liberated tubules were then collected byfiltration. The tubules were then dispersed into a solution of thetarget molecule. The solution was then filtered to remove the tubules aswell as the target molecule BQ sequestered inside.

FIG. 4 shows an example of the second method, dispersion of theliberated nanotubules. This figure shows first the UV absorptionspectrum of a solution that was 1×10⁻⁵ M 7,8 benzoquinoline solution(BQ). To this solution was first added silica nanotubules that did notcontain the hydrophobic C₁₈ silane inside. (10 mg of tubules added per100 mL of solution.) The solution was then filtered to remove thesetubules and the solution spectrum was remeasured. Note that there isessentially no change in the BQ absorbance. This experiment showed thatsilica tubules that were hydrophilic on the inside did not extract thehydrophobic BQ.

An identical quantity of the C₁₈ derivatized tubules was then added tothe solution. The solution was then filtered to remove the tubules andthe spectrum was remeasured. As indicated by the lower absorbance, thesetubules extracted 82% of the BQ from the solution. FIG. 5 showsanalogous data after addition of a second 10 mg of tubules per 100 mL ofsolution. After the second extraction 92% of the BQ was removed from thesolution.

In another embodiment of the invention, reactive molecules such asenzymes can be incorporated into nanoparticles to improve the drugdetoxification ability of the nanoparticles. Although enzymes aredescribed herein, incorporation of any molecule capable of generating achemical reaction or aiding in the rate of a chemical reaction with atarget molecule can also be incorporated in nanoparticles to produceenhanced detoxification results. This approach detoxifies substances andsurfaces by using nanoparticles as a platform to incorporate moleculessuch as enzymes for catalyzing the conversion of toxic substances intoinactive substances (or for generating chemical reactions with toxicsubstances).

Protein (i.e., enzyme) attachment to SiO₂ nanotubules was achieved using2 steps. A tubule wall was functionalized with an aldehyde-terminatedsilane. A protein was then coupled to the aldehyde through primary aminosites on the protein. The enzyme used, glucose oxidase (GOD), was thefirst protein tested. GOD effectively catalyzes the oxidation of glucoseto glucono-1,5-lactone. In the presence of glucono-1,5-lactone,electrons shuttle to O₂, creating hydrogen peroxide. In the presence ofperoxidase (POD), hydrogen peroxide oxidizes o-dianisidine from acolorless to red form, which then can be assayed by monitoring itsabsorbance (See FIG. 6).

For preliminary experiments performed, an intact alumina templatemembrane with the SiO₂/GOD nanotubules in the pores was utilized. Theabsorbance of a solution of glucose, o-dianisidine and POD immersed in a“blank membrane” (without nanotube incorporation of GOD) was determinedto establish a baseline absorbance. Al₂O₃/SiO₂/GOD membranes were thenimmersed into the glucose solution. The time dependent changes in theabsorbance (concentration) of glucose (assayed indirectly via oxidationof o-dianisidine) were determined. The results are depicted in FIG. 6.Based on the absorbance spectra shown, it is apparent that GODincorporated into pores of the nanotubules converted much of the glucosein the solution to glucono-1,5-lactone upon immersion of the membrane(at approximately 140 seconds). As shown in FIG. 6, at approximately 400seconds the membrane was removed from the solution. No further oxidationof glucose is observed because in removing the membrane the GODincorporated inside is removed.

The nanotubes are important because they are new morphologies ofparticulate material. They are also important, being newly available,for evaluation and use in biomedical applications either by themselvesor modified as described in this application. However, the nanotube isnot the only shape carrier/core particle that can be derivatized as isdescribed in this application. Many other shapes are useful, such asderivatized polyhedral-shaped porous (templated or not) nanoparticles.

Based on this observation, it was concluded that enzymes incorporatedinto nanoparticles can be used to degrade drugs. The linkage of enzymesto the inner surface of pores, can be achieved without losing theenzyme's reactivity. Although nanotubes having inner cavities for enzymeattachment was used, any shaped particle, whether tubular or not, havingpores adapted for this purpose can have enzymes inside the pores. Thus,any nanoparticles having pores, whether the pores are tubular or anyother shape may be used with the invention.

An identical approach (i.e., linking a cytochrome P-450 (CYP) enzymesystem) to the inner surface of a nanoparticle) can also be used toefficiently reduce the free concentration of lipophilic agents in humanplasma and blood by a biotransformation dominated mechanism.

Analogous experiments have been done with nanotubules that wereliberated from the template membrane, and substantially identicalresults were obtained. In this case the GOD was on both the inner andouter surfaces of the membrane. This is an advantage of the hollownanotubule approach. Having available inner and outer surfaces increasesthe surface area available for biocatalysis. As before, it might also beadvantageous to separately derivatize the inner and outer surfaces. Forexample, the inner surface could be derivatized with a specific dye andthe outer surface could be derivatized with a specific enzyme or otherprotein. In a second set of tubules, a second specific dye could beattached to the inside and a second specific protein to the outside.This could be continued for n tubules that contain specific dyes andspecific proteins on the insides and outsides. The dye could then beused to identify the tubules. For example, green tubules could containprotein #1, blue tubules could contain protein #2, etc. In this way, ina mixture of tubules one could identify which tubules are catalyzingwhich biochemical process.

Hollow and solid nanoparticles can also be used for removal oflipophilic toxins. For example, nanoparticles, whether hollow or solid,having substantially polyhedral or spherical morphologies can be usedfor this purpose. Studies were performed to establish whether thebenzene ring moiety of the prototypical amide local anesthetic,bupivacaine, possessed sufficient electron enrichment to enable π-πelectron bonding to an electron deficient molecular moiety attached to asolid nanoparticle. Specifically, when a mimic of bupivacaine was mixedwith the dinitrobenzamide moiety, a number of changes in spectral valuesoccurred. Specifically, the UV-VIS diffuse shoulder from 280-320 nmmoved to a diffuse shoulder from 340-400 nm.

Complexation of the pi-pi type between bupivacaine and its mimics, andseveral electron deficient aromatics, including a dinitrobenzamide(designed for convenient subsequent attachment to nanoparticles), hasbeen proven using proton NMR spectrometry. Tables 1 and 2 show thechemical shift values observed for test systems relevant to thisapplication. TABLE 1 Values of Shift for Donor-Trinitrobenzene Complexesin Chloroform-d Donor Shift (ppm, for acceptor) Direction2,6-Dimethylaniline 0.2995 Upfield 2,4-Dimethylaniline 0.3086 Upfield3,5-Dimethylaniline 0.2647 Upfield 3,4-Dimethoxytoluene 0.2730, 0.1 868bUpfieldnotes:a [donor:acceptor] = 60:1b at concentration in literature, lit.Value = 0.1830

TABLE 2 Values of Shift for Donor-N-Methyl-3,5-dinitrobenzamideComplexes in Chloroform-d Donor Shift (ppm, acceptor) Direction2,6-Dimethylaniline Triplet 0.0874 Upfield Doublet 0.0779 Upfield2,6-Dimethylace- Triplet 0.0156b, 0.1584 Upfield tanilide Doublet0.00775b, 0.0055 Downfield, Upfield Bupivacaine (salt) c Triplet 0.0891Upfield Doublet 0.0275 Upfieldnotes:a [Donor:Acceptor] = 60:1,except case b where [D:A] = 1:1;c Studied in 50:50 D20:CD3CN

This data is consistent with the ranges published (Dust, 1992) for otherπ complexed electron deficient and electron rich benzene rings. Thus,the dinitrobenzamide moiety can be attached to carrier nanoparticlesduring synthesis for use treating local anesthetic detoxificationprimarily by π-π complexation which can take place on solid or soft/hardnanoparticles, and with or without two-point binding or templated coresor shells.

Further experiments were performed to determine the effectiveness ofvarious emulsions to reduce the free concentration and toxic effects ofother drugs. As shown in FIGS. 7-9, the cardiotoxic effects ofbupivacaine, a local anesthetic, were significantly reduced using amacroemulsion of Intralipid.

FIG. 7 shows a macroemulsion of Intralipid. Intralipid attenuatesbupivacaine-induced sodium current (I_(ns)) in guinea pig ventricularmyocytes. Panel A shows examples of current traces in response todepolarization to −20 mV from a holding potential of −100 mV duringinterventions shown near cach records. Panel B shows the change incurrent peaking as a function of time. Horizontal bars above panel Bdenote the duration of drug administration. Panel C summarizes theeffect of bupivacaine (5, 10 and 20 μM) and Intralipid (1.5%) on I_(ns).All data was normalized to a control current. Bars represent themean±SEM of 5-7 myocytes.

FIG. 8 shows concentration-dependent in vitro attenuation of bupivacaine(1 μM). Bupivacaine induces QRS prolongation in guinea pig isolatedhearts, paced at 200 beats per minute (BPM) as shown in FIG. 8. FIG. 8shows the mean±SEM of 4 experiments (P<0.05). As shown in FIG. 8,increasing Intralipid concentations reduce QRS prolongation caused bybupivacaine 1%.

FIG. 9 shows the attenuation of in-vivo cardiotoxic effects ofbupivacaine in isofluanc-anesthetized rates. Specifically, the effect ofan IV bolus of bupivacaine (8 mg/kg over 10 scc) on the QRS interval isshown. Compared to time matched controls, Intralipid (3 ml/kg/min over 2min) more rapidly attenuated bupivacaine induced prolongation of the QRSinterval. Two additional experiments were also carried out, bothyielding similar results.

Therefore, compared to available methods, bioparticles which may beproduced using the invention have numerous advantages over currentmethods for treating drug toxicity. Advantages from the invention areenhanced through use of complementary approaches including lipidpartitioning, adsorption and xenobiotic biotransformation.

Bioparticles using lipid partitioning and/or drug biotransformationproduced using the invention not only scavenge most toxic drugs that aremore lipophilic (active drug state normally, but also offer broadersubstrate usage. For example, various soft bioparticles can effectivelyreduce the free blood concentration of all virtually lipophilic drugs.Moreover, appropriately chosen enzymes incorporated into bioparticlescan further improve the bioparticle's therapeutic performance andapplicability by adding metabolization effects applicable to a broadrange of drugs. If desired, the feature of chemical selectivity inherentin immunotoxicotherapy can be incorporated into bioparticles by usingthe processes of molecular templating and/or adsorption ontofunctionalized surfaces.

Using bioparticles, large lipid-water partition coefficients for highlylipid soluble substances such as amiodarone indicate that the freeconcentration of this antiarrhythmic agent can be effectively reduced byusing a concentration of soft bioparticles in the bloodstream thatshould not be detrimental to cell function (approximately 1.5% maximum).A bioparticle having a large lipid-water partition coefficient (e.g.10,000), where the lipid component of the bioparticle is either liquidsolid core, or lipophilic molecular entities attached to the surface ofan inorganic core can bind a large fraction of highly lipophilic drugs.Thus, a drug's free blood concentration can be effectively reduced in asmall volume of soft bioparticles.

In a preferred embodiment of the invention, bioparticles containing P450cytochrome components such as a CYP 3A4 fraction are used to not onlyoffer broad substrate detoxification, but also to produce rapidelimination of toxins from the blood. CYP3A4 and CYP2D6 hepaticmicrosomal fractions of the P450 system can biotransform approximately55% and 25%, respectively, of virtually all xenobiotics [Benet L Z,Kroetz D L and Sheiner L B, Pharmacokinetics in Goodman and Gilman's,Pharmacological Basis of Therapeutics (1996) eds Hardman J G and LimbirdL E, 9th Edition, McGraw Hill, pp 3-27.] Cytochrome results in anenhanced drug elimination rate by either increasing the quantity orquality of enzyme (e.g., selecting high activity enzyme systems usinggenetic polymorphisms or molecular cloning) and/or optimizing theenvironment of the enzyme (substrate concentration, cofactor levels,hydrophilicity for optimal enzyme functioning).

Bioparticles can provide partitioning and a biotransformation to takeadvantage of the potential synergistic actions which can result frominitially partitioning a drug at high local concentrations in anenvironment containing high concentrations of an enzyme. This synergycan dramatically increase the efficiency of the substrate degradation ifthe toxic drug concentration occurs well below the KM value.

For example, partitioning a toxic drug from the bloodstream into thelipid environment of a bioparticle containing a genetically engineeredP450 enzyme designed for super high efficiency (e.g., supersomes) shouldnot only effectively and promptly reduce the free blood concentration ofxenobiotics in the blood but also concentrate the toxic drug in an areaadjacent to the active enzyme. This can promote extremely efficientcatalysis and degradation of target molecules.

The beneficial effects of bioparticles formed using the invention areexpected to be greatly enhanced by concentrating a large enzyme mass,such as genetically engineering P450 fractions selected for super highactivity within the biocompatible particle. Preferably, the bioparticleshould have a large internal surface area whose efficiency of catalyzingits substrate to its metabolite is marked augmented by the concentratingeffect of the soft bioparticle component (e.g., exposes the enzyme to ahigh concentration of its substrate). In addition to its substrateconcentrating effect, another advantage gained by incorporating a lipidmatrix within the bioparticle is that this structure may dramaticallyincrease the intrinsic biocatalytic efficiency of an enzyme.Salt-immobilized hydrolytic and co-factor requiring enzymes(lyophilizates of enzyme in a salt matrix) in organic solvents have beenshown to have 100-3000 times more activity than that observed in aqueousmediums (U.S. Pat. No. 5,449,613 to Dordick, et al.). This technologyapplied to CYP fractions located within bioparticles can produceextremely efficient biocatalytical tools for drug detoxification,particularly when preferred high efficiently molecular cloned supersomesare used.

Numerous products can be produced from the invention including thosecomposed of multiple types of nanoparticles for in-vivo detoxificationof drugs and toxins from humans or animals. For example, nanoparticlescan be synthesized for attenuating acute cardiotoxic effects oftricyclic antidepressant drugs (e.g., amitriptyline). However, theinvention is not limited only to tricyclic antidepressants, butencompasses all drug classes that may cause toxicity. In addition,biological toxins (e.g., snake and insect envenomation) may also bedetoxified using the invention. This can ensure human and animal safetyand welfare. Furthermore, endogenous toxins produced during organdysfunction or failure (e.g., hepatic or renal failure) may also beremovable using the invention to create “circulating hepatocytes” or“circulation nephons.”

Another potential product which can be produced from the invention is aproduct for detoxification of poison warfare agents that are used formilitary purposes (e.g., nerve gas). It is noted that warfare agents maybe solids, liquids or gases. Warfare agents can cause massiveintoxification of substances such as acetylcholine or tissue necrosisfrom direct toxicity (e.g., mustard gases). Rapid and simultaneousremoval of both the warfare agent and molecules causing injury may proveto be effective therapy to mitigate the dangers of these weapons of massdestruction. For example, bioparticles produced using the inventioncould be used intravenously to reduce the concentration of both thetoxin (e.g., sarin) and acetylcholine. Alternatively, the invention canbe used for skin or metal decontamination for other types of toxicwarfare agents such as mustard gas.

1-20. (canceled)
 21. A method comprising: contacting a toxic compoundwith a composition comprising a particle comprising: (a) a first regioncomprising an enzyme, wherein the enzyme acts to transform the toxiccompound into a substantially inactive compound; and (b) a second regioncomprising a material selected to partition the toxic compound into thesecond region, wherein the first region is in contact with at least aportion of the second region, and wherein the contacting is for a timesufficient to allow the enzyme to transform a portion of the toxiccompound to the substantially inactive compound.
 22. A method of claim21 wherein the composition is contacted with the body of a subject. 23.The method of claim 22, wherein the subject is an animal.
 24. The methodof claim 22, wherein the subject is a human.
 25. The method of claim 22,wherein the contacting comprises intravenous delivery.
 26. The method ofclaim 22 wherein the particle comprises a hollow tube open at least atone end, wherein the hollow tube comprises an inorganic or polymermaterial and wherein the first region comprises a hydrophobic compoundattached to an inside surface of the hollow tube and the second regioncomprises the enzyme attached to a surface of the hollow tube.
 27. Themethod of claim 22, wherein the particle has a size from approximately 1to 200 nm.
 28. The method of claim 27, wherein the particle has a sizefrom approximately 1 to 5 nm.
 29. The method of claim 22 wherein thesecond region is an oil core and the first region comprises an enzymedispersed within the oil core and wherein a surface film encapsulatesthe oil core.
 30. The method of claim 22 wherein the second region is aliquid core and the first region comprises an enzyme dispersed withinthe liquid core and wherein an inorganic or polymer shell encapsulatesthe liquid core.
 31. The method of claim 22 wherein the first regionfurther comprises a coenzyme or a cofactor.
 32. The method of claim 22wherein the composition is contacted with the blood of a subject. 33.The method of claim 22 wherein the enzyme is a cytochrome P-450 enzyme.34. The method of claim 33 wherein the cytochrome P-450 enzyme isselected from a group consisting of a CYP3A4 and a CYP2D6 hepaticmicrosomal fraction.
 35. The method of claim 21 wherein the toxiccompound is selected from a group consisting of a drug, sarin, mustardgas, and a nerve gas.
 36. The method of claim 22 wherein the toxiccompound is a lipophilic drug.
 37. A method comprising: contacting atoxic compound with a composition comprising a particle comprising: (a)a core comprising an oil and an enzyme, wherein the enzyme is dispersedwithin the oil core and wherein the enzyme acts to transform the toxiccompound into a substantially inactive compound; and (b) a surface film,wherein the surface film encapsulates the oil core, wherein the oil coreis selected to partition the toxic compound into the second region, andwherein the contacting is for a time sufficient to allow the enzyme totransform a portion of the toxic compound to the substantially inactivecompound.
 38. The method of claim 37, wherein the surface film comprisesa surfactant.
 39. The method of claim 37, wherein the particle has asize from approximately 1 to 200 nm.
 40. The method of claim 39, whereinthe particle has a size from approximately 1 to 5 nm.